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Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9

Author

Listed:
  • G. Cullot

    (Bordeaux University, INSERM, BRIC, U1312)

  • J. Boutin

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

  • S. Fayet

    (Bordeaux University, INSERM, BRIC, U1312)

  • F. Prat

    (Bordeaux University, INSERM, BRIC, U1312)

  • J. Rosier

    (Bordeaux University, INSERM, BRIC, U1312)

  • D. Cappellen

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Tumor Biology and Tumor Bank Laboratory)

  • I. Lamrissi

    (Bordeaux University, INSERM, BRIC, U1312)

  • P. Pennamen

    (CHU de Bordeaux, department of medical genetics)

  • J. Bouron

    (CHU de Bordeaux, department of medical genetics)

  • S. Amintas

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Tumor Biology and Tumor Bank Laboratory)

  • C. Thibault

    (Bordeaux University, INSERM, BRIC, U1312)

  • I. Moranvillier

    (Bordeaux University, INSERM, BRIC, U1312)

  • E. Laharanne

    (CHU de Bordeaux, Tumor Biology and Tumor Bank Laboratory)

  • J. P. Merlio

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Tumor Biology and Tumor Bank Laboratory)

  • V. Guyonnet-Duperat

    (Bordeaux University, INSERM, BRIC, U1312
    Vect’UB, vectorology platform, INSERM US 005—CNRS UAR 3427-TBM-Core, Bordeaux university)

  • J. M. Blouin

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

  • E. Richard

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

  • S. Dabernat

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

  • F. Moreau-Gaudry

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

  • A. Bedel

    (Bordeaux University, INSERM, BRIC, U1312
    CHU de Bordeaux, Biochemistry Laboratory)

Abstract

The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.

Suggested Citation

  • G. Cullot & J. Boutin & S. Fayet & F. Prat & J. Rosier & D. Cappellen & I. Lamrissi & P. Pennamen & J. Bouron & S. Amintas & C. Thibault & I. Moranvillier & E. Laharanne & J. P. Merlio & V. Guyonnet-D, 2023. "Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-39632-w
    DOI: 10.1038/s41467-023-39632-w
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    References listed on IDEAS

    as
    1. Fatwa Adikusuma & Sandra Piltz & Mark A. Corbett & Michelle Turvey & Shaun R. McColl & Karla J. Helbig & Michael R. Beard & James Hughes & Richard T. Pomerantz & Paul Q. Thomas, 2018. "Large deletions induced by Cas9 cleavage," Nature, Nature, vol. 560(7717), pages 8-9, August.
    2. Grégoire Cullot & Julian Boutin & Jérôme Toutain & Florence Prat & Perrine Pennamen & Caroline Rooryck & Martin Teichmann & Emilie Rousseau & Isabelle Lamrissi-Garcia & Véronique Guyonnet-Duperat & Al, 2019. "CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations," Nature Communications, Nature, vol. 10(1), pages 1-14, December.
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